The production of transgenic cells and organisms is opening a very interesting field in genetic engineering; said field is making it possible to incorporate a large number of desirable characteristics into organisms that are directly or indirectly beneficial to man.
At the current state of the art, the methods that are used to control phytopathogenic fungi and nematodes involve the use of large amounts of chemicals, which make it possible to select strains that are resistant to them, making them unsuitable for use in the future, in addition to the fact that they remain in the soil for long periods of time and may prove toxic to both humans and to other species of animals and plants.
Because of this, the ecosystem may be irreversibly affected, which is always undesirable.
One alternative, which people have attempted to apply more widely, is to use agents that exist in nature and are the natural enemies of the phytopathogen(s) to be controlled. Many organisms that can be used in this way have been described in the state of the art. However, the use of said control agents has been limited, primarily because in the majority of the cases the level of control has not been comparable to that achieved with a very efficient chemical fungicide.
Among the most successful agents for biological control of phytopathogenic fungi are those belonging to the genus Trichoderma. These organisms have been favored because they are able to control a wide variety of phytopathogenic fungi that are of great importance to agriculture.
Despite the relative success with which these organisms have been employed, it still has not been possible to achieve the desired levels of disease control.
Various techniques have been used in attempts to obtain organisms of this genus that have improved efficiency as biological control agents; the improvement techniques utilized include that of mutagenesis of both the physical and the chemical types, and protoplast fusion.
Even though the production of strains that have been improved by these techniques has been described, one of the most serious problems that has been encountered is that in these cases some of the organism's desirable characteristics may be affected since it is not possible to direct the changes toward a single type of characteristic, at least as far as is now known in the prior art.
These techniques do not make it possible to modify these organisms selectively, nor do they guarantee that improved strains will be obtained.
A reliable and highly effective way of modifying these organisms without altering characteristics of theirs that are desirable, while still making it possible to do so in a controlled manner, is to introduce the desirable characteristic using the segment of genetic information that codes it in order to transform said organism.
The existing techniques do not make it possible to modify said organisms selectively, nor do they guarantee that improved strains will be obtained.
To date, the literature has given a detailed description of three methods of transforming the genus Trichoderma.
The first of these methods is based on forming protoplasts of this organism and subsequently treating them with polyethylene glycol (PEG) and calcium chloride (CaCl), in combination with thermal shock and in the presence of transformant DNA; this makes it possible to introduce said DNA into the cell being treated. Once inside the cell, the DNA is incorporated into the genome of the microorganism by a mechanism that is inherent in this organism itself (Herrera-Estrella et al., 1990, "High efficiency transformation system for the biocontrol agent Trichoderma spp.", Molecular Microbiology, 4:839-843).
The second method is based on using cells that are treated with a mixture of enzymes that are able to weaken or remove their cell walls; these cells are subsequently subjected for a brief period to an electric current that is generated with high voltage; this causes the transient formation of pores in the membranes of the cells being treated. If the cells are subjected to this electric shock in the presence of DNA, the DNA is introduced into the cell via the pores and is incorporated into the microorganism's genome in the same way as described above (Goldman et al., 1990, "Transformation of Trichoderma harzianum by high-voltage electric pulse." Current Genetics, 17:169-174).
Finally, the third method is based on accelerating solid and dense particles that are covered with the transformant DNA; these particles are made to strike the organism to be transformed, penetrating the organism's various coverings, and once inside the organism, the DNA dissolves and integrates into its genome by mechanisms that are inherent in it (Lorito et al., 1993, "Biolistic tranformation of Trichoderma harzianum and Gliocladium virent using plasmid and genomic DNA." Current Genetics, 24:349-356
These methods are known as transformation mediated by PEG (polyethylene glycol), electroporation, and bioballistics or bombardment, respectively.
All of these methods require that the cells recover from the treatment that they receive; this is normally accomplished by placing them in culture media that contain an osmo-protector, with the transformed cells being selected later. This is done by using markers, which can be fragments of genetic information that can express themselves in the receiving organism and can complement a requirement or auxotrophy of said organism, or they confer on said organism the ability to tolerate some antibiotic, or fungicide in this case.
As a result, for a long time attempts have been made to solve the above-mentioned problems in a simple and practical way, aiming at obtaining transformed organisms with improved biological control characteristics without affecting any other of the organism's characteristics, but to date this goal has not been achieved by a simple and easily implemented method.